1. Ch. 2A: How Do You Begin to Clone a Gene?

2. Learning goals Describe the characteristics of plasmids Explain how plasmids are used in cloning a gene Describe the function of restriction enzymes Explain how to use restriction enzymes to create a recombinant plasmid

3. Key Ideas Plasmids - ideal vectors for genetic engineering –replicate in the bacteria cell –gene promoter –antibiotic resistance as a selectable marker, –can be transferred into bacteria by conjugation. Restriction enzymes – key to the creation of a recombinant plasmid. –cut DNA at specific sequences –sticky ends allow strands to join

4. The Plasmid pARA-R plasmidplasmid

5. Recombinant plasmid of interest Bruce Wallace BamH I Hind III pARA-R 5,302 bp P BAD -rfp 806 bp pARA-R construct

6. sticky end BamH I sticky end Hind III sticky end BamH I sticky end Hind III 3’ 5’ 3’ 5’ 3’ Bruce Wallace Engineering the Plasmid: ligation of rfp gene into p-ARA

7. BamH I Hind III BamH I Hind III Restriction digest of pARA-R Recombinant plasmid of interest pARA-R 5,302 bp Biotech Experience P BAD -rfp 806 bp

8. Restriction analysis of pARA-R Restriction fragments after digest with Hind III and BamH I Biotech Experience 806 bp BamH IHind III BamH IHind III 4,496 bp

9. Learning goals: lab #4 Describe why it is important to verify products created in the genetic engineering process Predict the relative speed of DNA restriction fragments and plasmids through a gel during gel electrophoresis Separate and identify DNA restriction fragments and plasmids using gel electrophoresis

10. Key Ideas The multistep process that is used to clone a gene results in multiple products –Need to verify that you have the recombinant plasmid you need. DNA fragments and plasmids can be separated by gel electrophoresis. Loading dye helps monitor the progress of the gel electrophoresis procedure. DNA ladder helps determine the sizes of unknown pieces of DNA Gel is stained in order to show the location of the DNA fragments and plasmids.

11. Review questions 1.Why is it important to have sticky ends? 2.What is the purpose of the restriction enzymes? 3.How do you confirm the uptake of the gene into the plasmid? 1.Why is it important to have sticky ends? 2.What is the purpose of the restriction enzymes? 3.How do you confirm the uptake of the gene into the plasmid?

12. Clone That Gene activity 1. Cut the plasmid and the human DNA with the appropriate restriction enzyme 2. Insert the insulin gene into the plasmid DNA 3. Determine which antibiotic you would use to identify bacteria that have taken in the plasmid

13. Tips Reagents should be stored in a freezer until you are ready to prepare them for students. Allow to defrost for 15 minutes before using. The reagents can be aliquoted up to several days before the lab, then store in the freezer/refrigeratoraliquoted Vortex and spin enzyme mix and 2.5x RB before aliquoting video

14. Tips Calibrate and mark the controller “Floatie” marked with team number. Each period has a different color. Bottom of tubes in the water Low-tech water bath Multiple pans allow tubes from different classes to stay separated